Background: The effect of latitude on maternal and infant vitamin D status during lactation is presumed to be strongly associated with higher rates of deficiency in those living at higher latitudes, yet with lifestyle changes, this conclusion may no longer be correct. Objective: To ascertain if higher latitude adversely affects the vitamin D status of lactating women and their fully breastfeeding infants. Study Design/Methods: Fully breastfeeding women and their infants were eligible for participation in this study as part of a larger prospective vitamin D supplementation trial. Women were recruited from two sites of differing latitude: Charleston, SC at 32°N and Rochester, NY at latitude 43°N. Maternal and infant baseline vitamin D status, intact parathyroid hormone (IPTH), serum calcium and phosphorus as a function of site/latitude were measured. The primary outcome was maternal and infant total circulating 25(OH)D at baseline by center/latitude, and the secondary outcome was the percent of women and infants who had achieved a baseline concentration of at least 20 ng/mL, meeting the Institute of Medicine’s definition of sufficiency at 4 to 6 weeks postpartum. Statistical analysis was performed using SAS version 9.3. Results: Higher latitude adversely affected vitamin D status only in lactating Caucasian women. African American and Hispanic women and infants living in Rochester compared to Charleston had improved vitamin D status, an effect that was no longer significant when controlling for socioeconomic factors and season. Overall, therewas a significant vitamin D deficiency at baseline in lactating mothers, and a far greater deficiency in their infants. Maternal baseline 25(OH)D concentration remained positively associated with being Caucasian, BMI and summer months. Breastfeeding infant vitamin D status mirrored maternal status and remained positively associated with being Caucasian and summer months. Those infants who had been on a vitamin D supplement at the time of enrollment in the study had markedly improved vitamin D status compared to those infants not on supplement, but represented a significant minority of the cohort. Conclusions: There was a significant vitamin D deficiency among a cohort of women and their infants living at two diverse latitudes—Charleston, SC and Rochester, NY. Given the higher rate of vitamin D deficiency among African American and Hispanic women and their infants living at a lower latitude, conclusions about vitamin D status based on latitude alone may be faulty.